This SBIR project is designed to develop miniaturized cell-based biosensors for low cost, portability and enhanced shelf life by extending the viability of the cultured mammalian cells. We will demonstrate the benefit of our current technology by creating micro-chips with approximately 100 times smaller dimensions than the currently available Electrical Cell- Substrate Impedance Sensing (ECIS) systems. Because of the smaller chip dimensions there will be significant reduction in cost and media storage and replacement volumes. We will also optimize the media conditions by changing to appropriate CO2- independent buffer systems to extend the viability of the chosen mammalian cell line for a period of at least 45 days under ambient conditions. Moreover, we will incorporate multiparameter cellular sensing for improved toxicity detection over using a single parameter of cellular impedance. This small, low cost system with extended shelf life will have significant value as a portable field device for screening toxicants and pathogens in clinical settings, food and water supplies, as well as in high throughput applications for target validation in pharmaceutical industry. Specific aims of this SBIR proposal include: 1) Design and manufacture miniaturized circuits for impedance measurements, 2) Design and manufacture CO2- impervious microfluidic cards, 3) Propagate cells in CO2- independent media and monitor change in impedance during and after 45 days in a microfluidic environment, and 4) Evaluate toxicant responsiveness of the developed microfluidic card with at least three known chemical toxicants and compare with commercially available ECIS systems. PUBLIC HEALTH RELEVANCE: Cell-based biosensors (CBBs) have tremendous potential to be used as efficient, reliable and sensitive tools for the detection of pathogens, toxins or bioactive compounds for clinical, pharmaceutical, environmental, biosecurity and high-throughput screening (HTS) applications. Their early use in the screening of food and agriculture products would be beneficial as the safety of food, water and agriculture is of ultimate concern because of its significant impact on public health and economy. Mammalian cell-based biosensors are being developed for on-site use in clinics or hospitals as a first response in biodefense applications and for environmental monitoring. Currently these mammalian cell based systems are expensive because of their short shelf life, as mammalian cells are most difficult to keep in culture for several days without large external media delivery and maintenance systems. The low shelf life and high maintenance cost limits the use of an otherwise highly effective screening tool in field operations and in HTP screening applications. Therefore, significant advances are required to improve the present systems. The integration of microfabrication, microfluidics and cell culture have the ability to change the current scenario by fabricating sensors that are small, portable and cost effective because less raw material is required to fabricate them. In addition, the small size reduces the media and culture requirements. Moreover, the optimization of media conditions for field use would make them accessible to the user.